Cells have evolved intricate networks of interacting proteins that regulate gene expression in response to damage, external stress, or chemical signals. Regulated intramembrane proteolysis (RIP) is a highly conserved surveillance and response mechanism. The Ce envelope-stress response pathway is a RIP system found in Escherichia coli and many other Gram-negative bacteria. Under heat-shock conditions, outer membrane porins (OMP) misfold and accumulate in the periplasm. The newly exposed C-terminus of the misfolded OMPs binds to the PDZ domain of E. coli DegS and allosterically activates protease activity. The activated PDZ-protease serves as a molecular switch for the Ce response network, setting in motion a cascade of events that leads to the transcription of stress-response genes. The overreaching goal of this proposal is to expand our understanding of the mechanism of DegS allostery and regulation. Asymmetric DegS trimers with different combinations of active PDZ domains and/or protease domains will be constructed and characterized biochemically to interrogate the importance of symmetry in DegS activation and regulation. Key mutant proteins will be crystallized and their structures solved to aid in interpreting the biochemical data. The role of allosteric communication within and between DegS monomers will be examined. Structural analysis and sequence based statistical methods will be employed to identify residues that stabilize active or inactive DegS, and appropriate mutants will be constructed and characterized to understand the evolution of allostery within DegS. Finally, a fluorescence assay will be developed to allow rigorous determination of the kinetics and thermodynamics of DegS activation as a function of activating peptide, substrate, and environmental conditions. Preliminary results indicate that DegS labeled with a solvatochromic fluorophore changes fluorescence in an OMP-peptide dependent manner, setting the stage for these studies. In many of bacteria, Ce related gene expression is required for virulence. As DegS serves as the molecular gatekeeper for the envelope-stress response, it is an attractive target for antibiotic inhibition. Thus, understanding the allosteric mechanism that has evolved within this regulatory protease may lay a foundation for novel therapeutics. Finally, understanding the molecular determinants of DegS function may lead to the discovery of paradigms that are applicable to other PDZ-proteases including the human homolog HtrA2/Omi that has been linked to caspase-independent apoptosis and the prevention of cancer. PUBLIC HEALTH RELEVANCE: The pathogenicity and antibiotic resistance of many bacteria depends on their ability to detect and respond to extracellular stress. Therefore, understanding the molecular control mechanisms of the response systems is of importance to human health. The research described in this proposal will broaden and deepen our understanding of the allosteric mechanisms used by the DegS sensory protease to facilitate bacterial survival under inhospitable conditions.